The initially supported studies associated with this grant focused on the isolation and characterization of bioactive molecules which caused ectopic bone and cartilage formation when implanted into in vivo sites. This work emphasized the presence of progenitor mesenchymal cells which responded to these bioactive factors. The focus of the first subsequent renewal application was to characterize a preparation of progenitors (referred to collectively as "Mesenchymal Stem Cells"). It is suggested (by the applicant) that substantial progress has been made on the two Specific Aims of that proposal and this second competitive renewal application now focuses on one of these Aims, in which efforts were previously proposed to optimize cell-mediated repair of large femoral defects in rodents. The investigators now propose a multi-institutional collaboration to study the factors involved in engineering fracture repair. A 2 mm femoral gap in rodents will be analyzed for repair by inserting a uniform plug of cultured-expanded and marked marrow-derived "mesenchymal stem cells". It is suggested that this model allows a detailed analysis of this "pseudo repair blastema" and focuses on the cellular and molecular contributions of these cells to the repair tissue. Three Specific Aims are complimented by three separate experimental projects. In the first Aim, the applicants propose to determine the contribution of these stem cells to the repair process and to the repair tissue. In the second Aim, they propose to insert plugs which contain relatively homogenous populations of cells that are at three different stages of osteogenic lineage and to determine their contribution to the fracture repair tissue. The last Aim will focus on the contribution of mechanical load to this repair process and its effects on stem cells and the population of cells from the three distinct stages of osteogenic lineage. The goal of the proposed research is to test several predictions related to the control of engineered fracture repair at one well-defined site. The molecular, cellular and mechanical determinants involved in such an engineered fracture repair have considerable clinical complications and the proposed studies are designed to highlight the key and controlling features of these events.